Download Some effects of environment and hormone treatment on

254
W. W. SUHWABE:
[J.L.s.B. LVI
Some effects of environment and hormone treatment on reproductive morphogenesis
in the Chrysanthemum. By W. W, SOHWABE.Research Institute of Plant
Physiology, Imperial College, London
(With Plate 10 and 2 Text-figures)
INTRODUUTION
The study of morphogenesis in the plant is mainly concerned with the events at the growing points. I n the higher plants, in the vegetative condition, the active stem apices give
rise to the leaves and their associated lateral buds, and their initiation and development
follow a regular sequence which, under normal circumstances, is not easily disruptedexcept by surgical and other drastic techniques. The positioning of the young primordia,
their size at initiation relative to the main growing point and their rate of growth,
determine the phyllotactic pattern and also to some extent the general appearance of the
plant. When the plant becomes reproductive there is a change to an entirely different
sequence which is equally regular and fixed. I n contrast to the leaf, the flower
primordium represents an axial structure and in many instances it may be regarded as a
precocious lateral bud.
While the onset of reproduction frequently leads to the formation of modified leaves
or bracts before the actual flower primordia arise, this is only a very transitory phase and
each growing point must follow either one or other main sequence, and it is not possible
for it to persist in producing intermediate structures. This in itself indicates that when
reproduction starts, there is a complete switch-over at the apex from one morphogenetic
pathway to another. It is this fact which has also served to justify the concentration of
research on the actual ‘initiation’ of flowering; clearly it is ‘le premier pas qui coGte’.
Once reproductive development has begun, the growing point affected must either
continue this new line of development or f i s h ; only in relatively rare teratological
instances is a return to the earlier sequence possible. Experimentally, it is, of course,
possible to modify the various stages of floral or reproductivedevelopment also, but under
normal conditions this does not usually take place.
Confining attention to the Chrysanthemum, firat consideration is to be given to
what is known about the behaviour of the vegetative apex, which factors of the environment, etc., are known to affect the transition from the vegetative line of development to
the reproductive, and what changes have been observed to occur at the apex during this
stage. Finally, the factors influencing the later stages of reproductive development will
be discussed. It must be stressed, however, that in a plant of such horticultural interest
as the Chrysanthemum large numbers of different varieties have been selected-often
specifically for differences in their physiological behaviour. Hence, varietal differences
in response must be pronounced and not all the factors to be discussed will be of equal
importance in all strains. I n most of the experimental work to be described, the variety
‘Sunbeam’ was used, and it is believed that this variety is relatively unselected and
perhaps nearer to the ‘wild type’ of Chrysanthemum than many other varieties.
A. THE VEGETATIVE GROWING POINT
The vegetative growing point of the Chrysanthemum is seen in PI. 10, after all young
leaf primordia have been cut away. The bare apex, unoccupied by such primordia, is
approximately 1 6 0 , in
~ diameter, and its mean area remains relatively constant throughout the period of vegetative growth. During this period it cuts off leaf initials a t a regular
rate. At initiation the area of each primordium is approximately equal to the transverse
area of the bare apex itself as determined by Evington (1964),using the methods evolved
by Riahards (1948, 1981).
J.L.S.B. LVI]
REPRODUCTIVE MORPHOGENESIS I N THE CHRYSANTHEMUM
255
The rate of primordium formation during vegetative growth may vary from 2.2 to
3.2 per week, i.e. defining the rate of initiation at the apex as the 'true plastochron ' this
varies from 3.2 to 2.2 days. The variation is due to the fact that the previous history of
the plant and also the prevailing environment can affect this rate. The effects of a variety
of factors and their combinations have been investigated; one of the most important
being the temperature history of the plant, i.e. whether it has been vernalized or not.
Determinations of the 'true plastochron' for plants grown in identical conditions, but of
different temperature history yielded the following values :
Unchilled control
2 weeks chilling
4 weeks chilling
3-18f 0.18 days
2.44 0.13 days
2.26 f0.18 days
The actual growth temperature does not appear to affect the 'true plastochron' very
much, regardless of whether the temperature is held constant or whether different day and
night temperatures are given, as will be seen below from the data of two experiments:
Constant day and night
temperature
17' C., 2-79 d ~ y s
22" C., 2.54 days
27" C., 2.69 days
Day temperature
17" C., 2-25 days
22" C., 2.14 day8
27" C., 2-25 days
Night temperature
17"C., 2-29 days
22" c., 2.25 days
27" C., 2.10 days
This relative independence of temperature applies, however, only t o the 'true plestochron', i.e. the actual rate of leaf primordium initiation as determined by dissection of
the buds.
The 'apparent plastochron', i.e. the rate of unfolding of new leaves from the bud, as
determined by leaf counts, is affected very much more by the actual growth temperature,
and the following values were determined in one experiment :
18"C., 3.86 day6
22" C., 3.02 days k0-18
26" C., 2.61 days
The marked difference between these two measures is due to the fact that at lower
temperatures more leaves are accumulated in the terminal bud which then do not expand
as rapidly as they are initiated. Internally there is, of course, some heat production in
the bud, and the apex within it is also relatively well insulated by layers of air between
the primordia. Therefore, a mechanism retarding expansion of the outer primordia
might perhaps serve as a homoeostatic regulator allowing the growing point itself to be
a t a somewhat different temperature from the outer environment. However, this suggestion is purely speculative, and in fact it seems doubtful whether it could quantitatively
account for the observed effect.
Before leaving the discussion of the behaviour of the vegetative apex, brief reference
may be made to the fact that the morphology of the young primordia themselves is also
determined a t an early stage in the bud. The external environment is also capable of
modifying their final shape while acting at an early stage in their ontogeny. A good
example of this is seen in the temperature effect on leaf shape. I n the Chrysanthemum
the degree of dissection varies very much with the temperature prevailing during initiation. Leaves initiated at relatively high temperature (27' C.) become very much less
dissected than those formed at a lower temperature (17" C.). (Text-fig. 1.)
I
B.
THE CHANGE-OVER TO THE REPRODUCTIVE CONDITION
(i) Effects of the environment. In the Chrysanthemum, as in so many other plants, the
onset of reproductive development is largely subject to control by the environment.
The actual conditions required have been fairly closely studied and at least for the
Text-fig. 1. Temperature effect on leaf shape. Leaves arranged in order of their ages.
Left: from a plant grown at 27" C.; right: from a plant grown at 17' C.
..
J.L.S.B. LVI]
REPRODUCTIVE MORPEOQENESIS IN THE UHRYSANTHEMUM
257
variety ‘Sunbeam’ are now known in considerable detail. It must be stressed, however,
that other Chrysanthemum varieties are likely to show at least quantitative differences
from the optimum valves established.
For brevity’s sake, the various factors controlling the switch-over to the reproduction
condition and its continuation have been summarized in tabular form (Table l), which
also shows the effective ranges of duration and level.
Table 1. Sequence of factors controlling the initiation of reproductive development in
the Chrysanthemum,and their optimum levels and duration
Factor
Low temperature (vernalization)
Growth temperature
Day
Night
Long-day treatment
Short-day treatment
I
Light intensity during growth
period
(Variety ‘Sunbeam’)
Level
Duration
1°-120 c.
3-4 weeks
22”-23” C.
Until flowering Schwabe, Unpubl.
3-4 weeks
From then on
till flowering
> Approx. 1600-2000 f.c. Throughout
16 hr. day
8-10 hr. day
Reference
Schwabe, 1960
I
Schwabe, 1952a
Schwabe, 1962b
This table represents, of course, an oversimplification,since there are numerous interactions between the various factors. As regards their seat of action, it is also known that
those conditions involving the light factor, i.e. light intensity and duration, etc., are
perceived by the leaves. On the other hand, grafting experiments and experiments in
which different parts of the plants were subjected to different temperature conditionshave
shownthat the vernalizationeffectis perceived in thegrowingpoint itself.The physiological
changes wrought there persist only in the meristem and are not translocated about the
plant through mature tisaue, but they may increase in parallel with apical growth and
in this way be passed on to newly arising axillary meristems (Schwabe, 1954).
(ii) Internal and nutritional factors. Not much is known as yet about the internal
factors involved in the onset of flowering. Nitrogen content, as controlled by the level
of this nutrient in the culture medium, has only a slight effect by accelerating or, when
deficient, retarding growth generally. In one experimentnitrogen-deficientplants budded
a mere 4 days earlier than the controls with full nutrient supply, and the number of
leaves produced prior to bud formation was actually lower than in the controls. Also
chromatographic analysis of the growing points of vegetative and budded plants did not
reveal any marked differences in the free amino acids present.
Carbohydrate level also seems to be of little importance for the vernalization processes
accelerating flowering in this plant. This was revealed in some experiments on the devernalization of the Chrysanthemum. Such de-vernalization can be achieved by prolonged exposure to low light intensity treatment (Schwabe, 1957b), a treatment which
serves, of course, to deplete the carbohydrate reserves of the plant very severely indeed.
In plants so treated stem growth and leaf expansion are brought almost to a complete halt.
Nevertheless, such starved plants can be completely re-vernalized a t once by a second
chilling period while still in the starved condition. Also, while application of soluble
carbohydrate to such plants will permit stem growth and leaf expansion to continue, it
will not avert the de-vernalizing effect. Hence the role of carbohydrate in the vernalization process is at best subordinate.
(iii) Hormones. As regards hormones there is now some evidence that their application may delay or hasten the transition to the flowering condition. The initiation of
reproductive development may be delayed by indole-3-aceticacid in lanolin paste if this
is applied repeatedly just below the terminal growing point, even when the plant ia fully
vernalized. In one experiment six such applications of paste delayed bud formation by
258
W. W. SURWABE:
[J.L.s.B. LVI
three weeks and caused the auxin-treated plants to produce on the average an additional
six leaves before budding.
Gibberellic acid? on the other hand, applied in solution to the young leaves was shown
t o induce a proportion of plants to flower even in the absenoe of any chilling treatment,
as will be seen from Table 2, but the effect waa very slight, when leaf number figures are
considered, compared with that of vernalization. Other synthetic growth substances
auch as triiodobenzoic acid do not appear to affect the onset of the reproductive phase
to any significant extent.
(iv) Changes at the apex itself. One of the most striking changes at the apex during the
transition to the flowering state is the enormoua increase in the size of the bare, uncommitted apex itself. From a diameter of approximately 160p, in the vegetative condition,
the apex increases to approximately 3000p in diameter at the time just before floret
initiation, a 399-foldincrease in area. Although the apex increasesgradually during growth
most of this change in apical size occurs over the relatively short period of a few days.
Table 2. Effect of gibberellic acid on jlowering of vernalized and unvemzalized
Chy8anthmum plants
(Variety ‘ Sunbeam’. Ten replicates.)
Proportion
Treatment
budded
Unvernctlized:
0 p.p.m. gibberellic mid (control)
10 p.p.m. gibberellic mid (2 c.c.)
100 p.p.m. gibberellic mid (2 c.c.)
Vernalized :
0 p.p.m. gibberellio mid (control)
10 p.p.m. gibberellic mid (2 c.c.)
100 p.p.m. gibberellic mid (2 0.0.)
0/10
1/10
4/10
lop0
10/10
lop0
*Leaf number
to budding
- (37.0)
49
26.0
26.3
27.0
* Leaf number of vegetative plants at end of experiment in parenthewe.
Text-fig. 2. Transition from foliage leaves to inflorescence brmta.
Kindly donated by Dr P. W. Brian.
(38.8)
40.8 (35.1)
J.L.S.B. LVI]
REPRODUCTIVE MORPROUENESIS IN THE CHRYSANTHEMUM
259
Simultaneously, the other well-known changes take place ; the type of primordium initiated changes and after a small number of rather reduced leaves have been formed the
floral bracts areproduced (Text-fig.2). At this stage there is a large and in thechrysanthemum quite rapid rise in the order of phyllotaxis. Measured in Richards’s (1951) equivalent
phyllotaxis index, the index is about 2.1 for the vegetative apex, and rises by 1.8 units in
the reproductive. Also the time required to initiate these inflorescence bracts is reduced
very considerably. Whereas, as was seen above, vegetative plants require 2.2 and 3.2 days
in the vernalized and unvernalized state respectively for the initiation of each leaf, a
rough average plastochron for inflorescencebracts is approximately 9 hr. To estimate such
times is not very easy technically, but fortunately the terminal and upper lateral inflorescence buds of the Chrysanthemum form a more or less linear series of development,
the uppermost being the most advanced. If the top and bottom members of such a set
of three successive buds on a plant are dissected and the number of primordia in them
determined, then a good estimate is obtained of the number of primordia present in the
intermediate bud. Dissection of this bud after a further period of 1-2 days or even
hours yields an estimate of the number of primordia initiated in the interval.
In the variety ‘Sunbeam’ about thirty such bracts are formed. Their size, especially
their thickness, becomes progressively reduced, and the ‘blades ’ of the innermost bracts
are only two cells thick, any further reduction in the radial dimension would seem
impossible and under ordinary circumstances bract production then ceases abruptly and
floret production commences. It is an interesting fact, which may well be associated with
the great reduction of the radial dimension of these primordia, that these bracts which
arise round the extreme edge of the enlarging bare apex carry no lateral bud initials in
their axils at all.
C. THEREPRODUCTIVE UROWINU
POINT
The actual moment when the apex is to be regarded as definitely reproductive might be
defined differently according to the problem under consideration. However, in general
it would seem to be most satisfactory to define it as the point when any further development of it must be reproductive, i.e. when the change has become irreversible. In the
Chrysanthemum this is the moment when the apex has enlarged and the receptacle is
formed. The normal sequence after this concerns only the formation and development of
the florets and their ultimate functioning.
As the initiation of the florets themselves begins (i.e. primordia representing axial
structures) the large bare apical area is invaded and very soon used up altogether, thus
terminating the growth of the original growing point. The rate of such primordial initiation has also been estimated for the outer florets in the same manner as described above
for the bracts, though, of course, with a correspondingly greater margin of error. The
time for the initiation of each of these florets amounts to about 24 min. under approximatelyoptimum greenhouse conditions. At this rate about 6Ofloretsare formed in one day,
and since the inner florets are probably formed even faster, about 3-4 days will suffice
to initiate the usual 200-300 florets in the bud and conclude this phase. The equivalent
phyllotaxis index at this stage is approximately 7. An apex in the process of initiating
floret primordia is seen in PI. 10.
However, the behaviour of the growing point, even after receptacle formation, is still
subject to environmental control. Of the various factors investigated it appears that the
light factor is the most important-acting via the leaves and leafy bracts. Two aspects of
the light factor are of importance in this respect-the duration of photoperiod and also
the intensity, and in unfavourable light conditions the whole sequence of development
may be completely arrested. It seems most likely that these effects are indirect, acting
through modification of the auxin metabolism of the plant. There are two lines of evidence for this: (a) any treatment causing an increase in auxin level appears to inhibit
260
W. W. SCHWABE:
[J.L.s.B.
LVI
floral development; while (a) treatments causing a reduction of auxin level appear to
overcome the inhibition.
Very briefly the experimental evidence for this is as follows. Apices of plants forming
a receptacle in long-day conditions cease to develop any further after this point has been
reached, and no florets are formed ;the bare receptacle ends the existence of the growing
point concerned (Pl. 10). Below it the next 3-4 lateral buds then grow out vegetatively,
giving rise to new shoots which may again form abortive inflorescence buds. A further
set of vegetative laterals will grow out below these buds and the cycle may be repeated
again and again. Although the plant, as a whole, is thus prevented from becoming truly
reproductive, the terminal growing point itself cannot revert to the vegetative condition.
It appears that a t the moment the receptacle is formed the main apex loses its apical
dominance and Unless this can be re-established after floret initiation dominance is
assumed by the laterals below, which then stop further growth of the terminal apex.
Similarly plants which have reached the stage of receptacle formation in short-day
conditions may be inhibited from flowering by transfer to long day, but here the stage
of development reached at the time of transfer is important. The latest stage at which
long-day conditions can arrest the further development is that of gynoecium formation
in the marginal florets.
Although there is little positive evidence as yet which is really convincing, it does seem
that in long days higher auxin levels are attained.
In view ofthis, another treatment claimed t o lead to enhanced auxin effectswas applied
to plants which had budded in the favourable short-day conditions. This treatment
which consisted in a severe reduction of light intensity (to about 25 f.c.)while the plants
remained in short days, also caused the suppression of further growth of the developing
inflorescence in the same way as long days, and, moreover, the same limitation of
effectiveness as regards the stage of development reached a t the beginning of treatment
was observed. Buds which had passed this stage were not arrested.
Finally, actual application of hormones (indole-3-acetic acid in lanolin paste) just
below the inflorescence bud proved to be quite as effective as long days or reduced light
in stopping development, although here the effect was of course limited to the bud so
treated. Once again the stage of development reached was of importance and older buds
continued their development even in the presence of auxin.
Reduction of auxin level was achieved by transfer of plants from long days to short
days, which allowed the arrested inflorescencebuds to recommencetheir development and
to produce flowers, if they were still alive at this time. Also, removal of the main sources
of the excess auxin produced in long days, i.e. the young vegetative laterals growing out
below the bud, permitted further development of the terminal bud. This could be done
either by complete disbudding of all laterals while the plant was maintained in long days,
or by severing the arrested inflorescence bud itself, leaving on only a few bracts, and
re-rooting it in long days. In all these instances inflorescenceswere produced. There was
some evidence that the leaves or bracts still exerted in long days some slight inhibition,
since floral development was even more rapid if the plants were transferred to short
days in addition to the disbudding.
However, with these treatments some abnormalities and teratological effects became
apparent. Although the development of the terminal growing point as a whole remained
entirely reproductive, small parts of it produced a little vegetative growth before again
ending in an inflorescence. For instance, in such inflorescences the initiation of bracts
was not suddenly halted a t the periphery of the receptacle, but continued until the whole
of the original apex had become covered with florets. Most of such florets arose in the
axils of bracts, much a~ in other members of the Compositae not included in the subfamily Chrysantheminae (in some Classifications) of which the Chrysanthemum is the
type genus, and which is in fact characterized by the absence of such bracts.
Another such observation was the appearance of secondary inflorescences from the
W. W. SCHWABE
Journ. Linn. SOC. Bot. Voi. LVI, Pi. 10
(Fucimg p . 261)
J.L.S.B. LVI]
REPRODUUTIVE MORPHOOENESIS IN TIIE CHRYSANTHEMUM
261
gynoecium of the marginal ligulate florets-like the hen-and-chicken daisy. A very
similar effect in Helenium, which was recently brought to notice, was almost certainly
due t o accidental spraying with a hormone weed killer.
Even the individual florets may be modified in their morphology. Thus hormone treatment of the main bud frequently leads t o the production of tubular instead of ligulate
florets in adjacent inflorescences (Pl. 10). Some varieties of the Chrysanthemum produce
such ‘quill’ flowers quite normally, and it seems possible that these may differ from other
varieties in their hormone levels at the susceptible stage.
I n concluding this very brief survey of the morphogenetic activity of the Chrysanthemum apex, it may be said that there are two sequences of organ production: the
vegetative, which is indeterminate and concerned with essentially similar structures
throughout, and the reproductive, which so far as the individual apex is concerned, is
determinate and is concerned with a sequence of structures showing a range of form.
Both sequences in themselves are relatively stable and can be upset by drastic treatments only, such as application of hormones or surgical techniques. The change-over
from one to the other condition is itself controlled by environmental factors and in the
Chrysanthemum, a t least, the change-over appears possible only in one direction. Once
the growing point has become reproductive it must complete this sequence or die prematurely. The actual change itself also follows a regular sequence. The great increase in
apical size is associated with a related change in primordial size and possibly as a consequence their h a 1 shapes. When the radial diameter of the primordia is so small that no
more bracts are produced, itself possibly a consequence of spatial factors, the bare apex
is rapidly used up in the formation of the florets.
It seems likely that in the Chrysanthemum, as in other plants, the change is controlled
by hormones and probably a balanced system of promotion and inhibition is concerned.
REFERENCES
EVINOTON,
D. G., 1954. A n investigation of Phyllotaxis end Apical Growth in Flax and Chrysanthemum. Ph.D. Thesis, London.
RICHARDS,
F. J., 1948. The geometry of phyllotaxis and its origin. Symp. SOC.
Exp. Biol. 2, 217-45.
RICHARDS,
F. J., 1951. Phyllotaxis: its quantitative expression and relation to growth in the apex.
Ph4. Trans. B, 235, 509-64.
SCHWABE,
W. W., 1950. Factors controlling flowering in the Chrysanthemum. I. The effects of
photoperiod and temporary chilling. J. Exp. Bot. 1, 329-43.
SCHWABE,
W. W., 1952a. Effects of temperature, day length and light intensity in the control of
flowering in the Chrysanthemum. Rep. X I I I t h Int. Hort. Congr. pp. 1-9.
SCHWABE,
W. W., 1952b. Factors controlling flowering in the Chrysanthemum. 111. Favourable
effects of limited periods of long-day on inflorescence initiation. J. Ezp. Bot. 3, 430-6.
SCHWABE,
W. W., 1954. Factors controlling flowering in the Chrysanthemum. IV. The site of
vernalization and translocation of the stimulus. J. Exp. Bot. 5 , 389-400.
SCHWABE,
W. W., 19570. The study of plant development in controlled environments. I n Control of
tke Plant Environment (5. P. Hudson, ed.), pp. 16-35. London.
SCHWABE,
W. W., 1957b. Factors controlling flowering in the Chrysanthemum. VI. De-vernalization
by low-light intensity in relation to temperature and carbohydrate supply. J . Exp. Bot. 8,
220-34.
EXPLANATION O F PLATE
PLATE10
(a) Bare apex (A) of vegetative Chrysanthemum plant after all leaf primordia have been cut away,
except the youngest ( P ) .
(b) Receptacle, formed in short day, in process of floret initiation.
(c)
Receptacle, formed in long day, and prested without floret formation.
(d) Abnormal production of tubular florets instead of ligulate florets, caused by application of
indole-3-acetic acid to the plant. Left : normal inflorescences; right : inflorescences with tubular
florets.